This invention relates generally to luminescent assay procedures, and, more particularly, to the suppression of extraneous light in luminescent specific binding assays.
Specific binding assays provide an economical means for detecting and measuring an analyte or ligand present in small concentrations in a sample. Specific binding assays are based upon the interaction of two bindable substances, one the analyte and the other a specific binding partner, which specifically recognize each other. Examples of specific binding partners whose interaction can serve as the basis for a specific binding assay include antigens-antibodies, biotinavidin, DNA probes, enzymes-substrates, enzymes-inhibitors, enzymes-cofactors, chelators-chelates, and cell surface receptor pairs. Assays involving other specifically bindable substances are also known and within the scope of the present invention.
While many variations have been proposed, one such assay involves binding the analyte in a sample with a specific binding partner previously conjugated with a component of a directly measurable labelling or tagging reaction. The labelling reaction is measured to determine the extent of the binding between the analyte and the conjugated specific binding partner, which, depending upon the particulars of the assay procedure, can reflect the amount of the analyte in the sample. Specific binding assays have shown great utility in determining various ligands in biological, medical, environmental and industrial applications.
A variety of labelling reactions have been proposed for use in specific binding assays, including radioactive, chromogenic and luminogenic procedures. In a radioactive labelling procedure, the component conjugated with the specific binding partner is an atom or molecule which emits radioactivity. Chromogenic and luminogenic labelling reactions are chemically more complex, in that several reactants may be involved. In one type of chromogenic or luminogenic reaction, a molecule changes color or emits light in a catalyzed reaction. The component conjugated to the specific binding partner can therefore be either one of the reactants, termed a substrate, or the catalyst. The remaining components of the reaction, that is, those not conjugated to the binding partner, are supplied in a chromogenic or luminogenic reagent medium, so that the uniting of the labelled conjugate and the reagent medium results in the desired color change or light emission, respectively.
A variety of assays using the principles of the specific binding approach are known, and several have become important diagnostic tools. In one such type of specific binding assay, the immunoassay, the analyte is an antibody, antigen, or hapten, and is made to react with another member of this group. The remaining background discussion will focus on such immunoassays, although this focus is made for clarity of presentation, and is not to be interpreted as limiting of the invention, which is broadly applicable to luminescently labelled specific binding assays.
Detecting the presence of reactive components in the body is a medically important diagnostic technique particularly suited to use of specific binding assays. In one important example, the immune reaction, the human body responds to certain foreign molecules called antigens by itself producing antibodies specific to the antigens, which help counteract the invading antigens. Some persons develop exaggerated responses to even small amounts of certain antigens. These reactions, which may be severe or even fatal, are termed allergic reactions. It is therefore highly desirable to be able to determine whether a person has allergies, and, if so, to what antigens, so that exposure can be avoided or so that the person may be desensitized to the antigen.
In the past, allergic sensitivity was measured by in vivo patch or stratch tests, wherein a possible antigenic irritant is contacted to the person's skin for a period of time, and the sensitivity judged by observing the skin reaction. As well as being imprecise and difficult to quantitate, such in vivo tests are expensive, inconvenient and time consuming for both physician and patient.
In vitro immunoassays can indicate the allergic condition of a person by determining the level of antibodies specific to certain antigens that are present in a fluid sample drawn from the patient's body. In one specific binding immunoassay for detecting the presence in the human body of antibodies to particular antigens, the antigen is attached to a solid surface designated for measurement and then exposed to human blood serum drawn from the patient, which may or may not contain the antibodies specific to the antigen. If antibodies are present in the serum, they react with the antigen and are thus also immobilized at the solid surface. The serum is removed, and the antigen-antibody pair, if present, is labelled by one of several means. For example, the antigen-antibody pair may be labelled with a radioactive atom conjugated to an anti-human antibody, so that the radioactive atom becomes immobilized at the surface (through the antigen-antibody pair) for subsequent measurement, only if the antibody analyte is present in the serum. This procedure, termed a radioimmunoassay, is effective but has certain drawbacks such as the need to use radioactive reagents which must later be disposed of, and limited sensitivity to low levels of analyte.
It has also been proposed to label the antigen-antibody pair attached to the surface with an anti-human antibody conjugated with one component of a luminogenic reaction system, the procedure being termed a luminescent immunoassay or LIA. The remaining components of the luminescent reaction system are provided in a subsequently introduced reagent medium, so that contacting the reagent medium to the immobilized labelled conjugate results in light emission. The light source for the luminescent assay may be chemical or biological in nature, so that the luminescent assays are respectively termed chemiluminescent and bioluminescent. The light emission in the luminescent assay may also arise from fluorescence or phosphorescence. The light emitted in any of these assays may be detected for measurement by any suitable means, such as, for example, a photomultiplier tube or photographic film.
Several problems arise in the use of luminescent specific binding assays. Of particular concern is the emission of light from a surface or volume other than that which is designated for measurement. Such light, termed extraneous light, interferes with the measurement of the light emitted from the designated surface. In the assay procedure just described, an undesirable extraneous lighting is produced adjacent the solid surface at which the tagged antigen-antibody pairs are immobilized, producing a "halo" effect around this designated surface during luminescence. This extraneous light broadens the apparent image of the surface and reduces its sharpness. The apparent relative intensity of the light emitted from the designated surface is altered as a result of this extraneous lighting. That is, a relatively weakly emitting area of a solid surface may appear to be somewhat brighter relative to the background than it actually is due to the extraneous light, with an apparent relative strengthening and broadening of its image. Inaccuracies of measurement can then result.
A related problem results from nonspecific binding. Although the described specific binding assays are generally highly specific, there may be nonspecific binding wherein a luminescent reaction component becomes immobilized at a surface even though no corresponding specific binding partner is present. If such nonspecific binding occurs, then nonspecific luminescence can be emitted, resulting in a false positive indication of reactivity even in an absence of corresponding analyte in the sample.
In another type of luminescent specific binding assay, an analyte or analyte analog is preferentially concentrated at a designated measurement surface, such as the bottom of a transparent tube wall. A first solution containing the analyte is added before or simultaneously with a specific binding partner for the analyte, conjugated with one component of a luminescent reaction, so that specific binding pairs are formed in solution and at the designated measurement surface. The remaining components of the luminescent reaction are then added in a second solution. However, reactive pairs found at other surfaces and throughout the tube volume, produce extraneous light emission, which can interfere with the measurement of light intensity from those pairs located at the designated measurement surface. One way to reduce such extraneous light is to physically remove the sample and the first solution from the tube before adding the second solution, but this approach necessitates a separation step. The assay thus requires a heterogeneous procedure rather than a homogeneous procedure, thereby increasing the cost of performing the assay. It would be highly desirable to avoid the interference from extraneous light in these circumstances so that the assay may be conducted homogeneously, particularly where many such tests are routinely performed and the separation step accounts for a significant cost.
Accordingly, there has been a need for an approach to suppressing undesirable extraneous light in luminescent assays. The present invention fulfills this need, and further provides related advantages.